Therapeutic multispecific polypeptides activated by polypeptide chain exchange

Abstract

The present invention relates to a set of heterodimeric polypeptides and its uses in therapy, e.g. for treating cancer.

1 . A set of heterodimeric precursor polypeptides comprising:
 a first heterodimeric precursor polypeptide comprising at least two polypeptide chains comprising a CH3 domain, wherein the two polypeptide chains comprising the CH3 domain are associated with each other via the CH3 domains and form a heterodimer, wherein one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation,   wherein the first heterodimeric precursor polypeptide comprises a first antigen binding moiety, wherein at least a part of the first antigen binding moiety is arranged on one of the two polypeptide chains comprising the CH3 domain, and   a second heterodimeric precursor polypeptide comprising at least two polypeptide chains comprising a CH3 domain, wherein the two polypeptide chains comprising the CH3 domain are associated with each other via the CH3 domains and form a heterodimer, wherein one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation,   wherein the second heterodimeric precursor polypeptide comprises a second antigen binding moiety, wherein at least a part of the second antigen binding moiety is arranged on one of the two polypeptide chains comprising the CH3 domain;   wherein   A) either i) within the first heterodimeric precursor polypeptide the polypeptide chain comprising the CH3 domain comprising the knob mutation comprises at least a part of the first antigen binding moiety and within the second heterodimeric precursor polypeptide the polypeptide chain comprising the CH3 domain with the hole mutation comprises at least a part of the second antigen binding moiety, or ii) within the first heterodimeric precursor polypeptide the polypeptide chain comprising the CH3 domain comprising the hole mutation comprises at least a part of the first antigen binding moiety and within the second heterodimeric precursor polypeptide the polypeptide chain comprising the CH3 domain with the knob mutation comprises at least a part of the second antigen binding moiety; and wherein   B) either i) the first heterodimeric precursor polypeptide comprises one polypeptide chain comprising a VL domain and the CH3 domain, and wherein the second heterodimeric precursor polypeptide comprises one polypeptide chain comprising a VH domain and the CH3 domain, wherein said VL domain and said VH domain specifically bind to an antigen when associated to a pair of a VH domain and a VL domain; or ii) the first heterodimeric precursor polypeptide comprises one polypeptide chain comprising a VH domain and the CH3 domain, and wherein the second heterodimeric precursor polypeptide comprises one polypeptide chain comprising a VL domain and the CH3 domain, wherein said VL domain and said VH domain specifically bind to an antigen when associated to a pair of a VH domain and a VL domain; and wherein   C) either   i) the CH3 domain of the first heterodimeric precursor polypeptide comprising the knob mutation and the CH3 domain of the second heterodimeric precursor polypeptide comprising the hole mutation, or   ii) the CH3 domain of the first heterodimeric precursor polypeptide comprising the hole mutation and the CH3 domain of the second heterodimeric precursor polypeptide comprising the knob mutation comprise the following amino acid substitutions, wherein the numbering is according to the Kabat numbering system:   the CH3 domain with the hole mutation comprises at least one amino acid substitution selected from the group of:
 replacement of E357 with a positively charged amino acid; 
 replacement of S364 with a hydrophobic amino acid; 
 replacement of A368 with a hydrophobic amino acid; and 
 replacement of V407 with a hydrophobic amino acid; and 
   the CH3 domain with the knob mutation comprises at least one amino acid substitution selected from the group of:
 replacement of K370 with a negatively charged amino acid; 
 replacement of K370 with a negatively charged amino acid, and replacement of K439 with a negatively charged amino acid; 
 replacement of K392 with a negatively charged amino acid; and 
 replacement of V397 with a hydrophobic amino acid. 
   
     
     
         2 . The set of heterodimeric polypeptides according to  claim 1 , wherein the CH3 domain comprising the knob mutation and the CH3 domain comprising the hole mutation indicated in C) comprise one of the amino acid substitutions selected from the group indicated in the following table: 
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   CH3 domain comprising hole 
                   CH3 domain comprising 
                 
                     
                   mutation 
                   knob mutation 
                 
                     
                     
                 
                     
                   E357K 
                   V397Y; K370E; K392D; or 
                 
                     
                     
                   double mutation K370E 
                 
                     
                     
                   K439E 
                 
                     
                   V407Y 
                   no mutation; V397Y; or 
                 
                     
                     
                   K370E 
                 
                     
                   S364L 
                   no mutation; V397Y; or 
                 
                     
                     
                   K370E 
                 
                     
                   A368F 
                   V397Y; K370E; K392D; or 
                 
                     
                     
                   double mutation K370E 
                 
                     
                     
                   K439E 
                 
                     
                     
                 
             
                
                
                
                
               
               
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         3 . The set of heterodimeric polypeptides according to  claim 1 , wherein the CH3 domain comprising the knob mutation and the CH3 domain comprising the hole mutation indicated in C) comprise one of the amino acid substitutions selected from the group indicated in the following table: 
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   CH3 domain comprising hole 
                   CH3 domain comprising 
                 
                     
                   mutation 
                   knob mutation 
                 
                     
                     
                 
                     
                   E357K 
                   V397Y; K370E; K392D; or 
                 
                     
                     
                   double mutation K370E 
                 
                     
                     
                   K439E 
                 
                     
                   V407Y 
                   V397Y; or K370E 
                 
                     
                   S364L 
                   V397Y; or K370E 
                 
                     
                     
                 
             
                
                
                
                
               
               
                
                
                
                
                
                
               
            
           
         
       
     
     
         4 . The set of heterodimeric polypeptides according to one of the preceding claims, wherein in case the CH3 domain with the knob mutation indicated in C) comprises a mutation E357K, the CH3 domain with the hole mutation indicated in C) does not comprise a mutation K370E. 
     
     
         5 . The set of heterodimeric polypeptides according to one of the preceding claims, wherein either i) the CH3 domain comprising the knob mutation of the first heterodimeric precursor polypeptide comprises a cysteine mutation and the CH3 domain comprising the hole mutation of the second heterodimeric precursor polypeptide comprises a cysteine mutation, or ii) the CH3 domain comprising the hole mutation of the first heterodimeric precursor polypeptide comprises a cysteine mutation and the CH3 domain comprising the knob mutation of the second heterodimeric precursor polypeptide comprises a cysteine mutation. 
     
     
         6 . The set of heterodimeric polypeptides according to one of the preceding claims, wherein the first antigen binding moiety and/or the second antigen binding moiety is an antibody fragment. 
     
     
         7 . The set of heterodimeric polypeptides according to one of the preceding claims, wherein
 a) the first heterodimeric precursor polypeptide comprises:
 a first heavy chain polypeptide comprising from N- to C-terminal direction a first VH domain, a CH1 domain, a second antibody variable domain selected from a VH domain and a VL domain, and a CH3 domain, 
 a second heavy chain polypeptide comprising from N- to C-terminal direction an antibody variable domain capable of associating with the second antibody variable domain of the first heavy chain polypeptide, and a CH3 domain, wherein the first heavy chain polypeptide and the second heavy chain polypeptide are associated with each other via the CH3 domains and form a heterodimer, wherein one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; and 
 a light chain polypeptide comprising from N- to C-terminal direction a first VL domain and a CL domain, wherein the first VH domain and the first VL domain are associated with each other and form an antigen binding site specifically binding to a target antigen; and wherein 
   b) the second heterodimeric precursor polypeptide comprises:
 a third heavy chain polypeptide comprising from N- to C-terminal direction a second VH domain, a CH1 domain, a third antibody variable domain selected from a VH domain and a VL domain, and a CH3 domain, 
 a fourth heavy chain polypeptide comprising from N- to C-terminal direction an antibody variable domain capable of associating with the third antibody variable domain of the third heavy chain polypeptide, and a CH3 domain, wherein the third heavy chain polypeptide and the fourth heavy chain polypeptide are associated with each other via the CH3 domains and form a heterodimer, wherein one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; and 
 a light chain polypeptide comprising from N- to C-terminal direction a second VL domain and a CL domain, wherein the second VH domain and the second VL domain are associated with each other and form an antigen binding site specifically binding to a target antigen; and wherein 
   c) either i) the first heavy chain polypeptide comprises a CH3 domain comprising a knob mutation and the third heavy chain polypeptide comprises a CH3 domain comprising a hole mutation; or ii) the first heavy chain polypeptide comprises a CH3 domain comprising a hole mutation and the third heavy chain polypeptide comprises a CH3 domain comprising a knob mutation; and wherein   d) the variable domains of the first heavy chain polypeptide and the third heavy chain polypeptide are capable of forming an antigen binding site specifically binding to a target antigen.   
     
     
         8 . The set of heterodimeric polypeptides according to one of the preceding claims, wherein
 a) the first heterodimeric precursor polypeptide comprises:
 a first heavy chain polypeptide comprising from N- to C-terminal direction a first VH domain, a CH1 domain, a second antibody variable domain selected from a VH domain and a VL domain, a CH2 domain and a CH3 domain, 
 a second heavy chain polypeptide comprising from N- to C-terminal direction an antibody variable domain capable of associating with the second antibody variable domain of the first heavy chain polypeptide, a CH2 domain and a CH3 domain, wherein the first heavy chain polypeptide and the second heavy chain polypeptide are associated with each other via the CH3 domains and form a heterodimer, wherein one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; and 
 a light chain polypeptide comprising from N- to C-terminal direction a first VL domain and a CL domain, wherein the first VH domain and the first VL domain are associated with each other and form an antigen binding site specifically binding to a target antigen; and wherein 
   b) the second heterodimeric precursor polypeptide comprises:
 a third heavy chain polypeptide comprising from N- to C-terminal direction a second VH domain, a CH1 domain, a third antibody variable domain selected from a VH domain and a VL domain, a CH2 domain and a CH3 domain, 
 a fourth heavy chain polypeptide comprising from N- to C-terminal direction an antibody variable domain capable of associating with the third antibody variable domain of the third heavy chain polypeptide, a CH2 domain and a CH3 domain, wherein the third heavy chain polypeptide and the fourth heavy chain polypeptide are associated with each other via the CH3 domains and form a heterodimer, wherein one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; and 
 a light chain polypeptide comprising from N- to C-terminal direction a second VL domain and a CL domain, wherein the second VH domain and the second VL domain are associated with each other and form an antigen binding site specifically binding to a target antigen; and wherein 
   c) either i) the first heavy chain polypeptide comprises a CH3 domain comprising a knob mutation and the third heavy chain polypeptide comprises a CH3 domain comprising a hole mutation; or ii) the first heavy chain polypeptide comprises a CH3 domain comprising a hole mutation and the third heavy chain polypeptide comprises a CH3 domain comprising a knob mutation; and wherein   d) the variable domains of the first heavy chain polypeptide and the third heavy chain polypeptide are capable of forming an antigen binding site specifically binding to a target antigen.   
     
     
         9 . The set of heterodimeric precursor polypeptides according to one of the preceding claims, wherein the VH domain and the VL domain indicated in B) are capable of forming an antigen binding site specifically binding to CD3. 
     
     
         10 . The set of heterodimeric precursor polypeptides according to one of the preceding claims, wherein no interchain disulfide bond is formed between the two polypeptide chains comprising the CH3 domains of the first and second heterodimeric polypeptide. 
     
     
         11 . A method for generating a heterodimeric polypeptide comprising contacting a first heterodimeric precursor polypeptide and a second heterodimeric precursor polypeptide, as defined in one of  claims 1  to  10  to form a third heterodimeric polypeptide comprising at least one polypeptide chain comprising a CH3 domain from the first heterodimeric precursor polypeptide and at least one polypeptide chain comprising a CH3 domain from the second heterodimeric polypeptide. 
     
     
         12 . The method of one of  claim 11 , wherein the second heterodimeric precursor polypeptide comprises an antigen binding moiety specifically binding to a second antigen, and wherein the third heterodimeric polypeptide comprises the antigen binding moiety specifically binding to the first antigen and the antigen binding moiety specifically binding to the second antigen, and a third antigen binding moiety is formed by the VL domain and the VH domain indicated in B). 
     
     
         13 . The method according to one of  claim 11  or  12 , wherein no interchain disulfide bond is formed between the two polypeptide chains comprising the CH3 domains of the first and second heterodimeric polypeptide, and wherein the contacting is performed in absence of a reducing agent. 
     
     
         14 . A heterodimeric polypeptide obtained by a method according to any one of  claims 11  to  13 . 
     
     
         15 . A first heterodimeric precursor polypeptide as defined in any one of  claims 1  to  10 . 
     
     
         16 . A second heterodimeric precursor polypeptide as defined in any one of  claims 1  to  10 . 
     
     
         17 . The set of heterodimeric precursor polypeptides according to any one of  claims 1  to  10 , wherein in the first and second heterodimeric precursor polypeptide the VH domain and the VL domain indicated in B) are capable of forming an antigen binding site specifically binding to CD3 for use in the treatment of cancer.